Magnus Edbladh

Effects of extracellular matrix components on axonal outgrowth from peripheral nerves of adult animals in vitro

Relatively little is known of the growth requirements for regenerating axons of the peripheral nervous system of adult animals. In the present study, we show that extracellular matrix material secreted by the Engelbreth-Holm-Swarm tumor cell line (matrigel) supports axonal growth from explanted peripheral nerve-dorsal root ganglia (DRG) preparations of adult mice and amphibia in serum-free media, without addition of growth factors. Axonal growth in matrigel was much more profuse than that in the more commonly used gels of type 1 collagen and, after some days in culture, was accompanied by migration of Schwann cells along axons. The most abundant protein in matrigel is laminin, which has been shown in many studies to support axonal growth but, surprisingly, antisera to laminin did not inhibit axonal growth in matrigel. To determine the ability of the major components of matrigel, laminin, type IV collagen, and heparan sulfate proteoglycan (HSPG), to support axonal growth, these proteins were added to preparations of mouse peripheral nerve-DRGs in type I collagen gels. Regenerating axons were significantly longer in the presence of laminin and type IV collagen than in control cultures, while HSPG had a slight inhibitory effect. In this assay system, however, diluted matrigel solution was even more effective in stimulating axonal growth than laminin or type IV collagen, either alone or in combination. The results suggest that in addition to laminin and type IV collagen, other components within matrigel may contribute to its ability to support axonal growth.

Insulin and IGF-II, but not IGF-I, stimulate the in vitro regeneration of adult frog sciatic sensory axons

We used the in vitro regenerating frog sciatic nerve to look for effects of insulin and insulin-like growth factors I and II (IGF-I, IGF-II) on regeneration of sensory axons and on injury induced support cell proliferation in the outgrowth region. In nerves cultured for 11 days, a physiological dose (10 ng/ml, approximately 2 nM) of insulin or IGF-II increased ganglionic protein synthesis (by 20% and 50%, respectively) as well as the level of newly formed, radiolabelled axonal material distal to a crush injury (both by 80%), compared to untreated, paired controls. In addition, insulin increased the outgrowth distance of the furthest regenerating sensory axons by 10%. The preparation was particularly sensitive to insulin during the first 5 days of culturing. Furthermore, both insulin and IGF-II were found to inhibit proliferation of support cells in the outgrowth region in a manner suggesting effects via their individual receptors. The inhibition, about 30%, was observable after 4 but not 11 days in culture. It is not clear if this reflects a stimulated differentiation of some cells. By contrast, IGF-I lacked effects on both regeneration and proliferation. In conclusion, the results suggest that insulin and IGF-II are involved in the regulation of peripheral nerve regeneration.

Protein kinase C and mouse sciatic nerve regeneration.

We have studied the role of protein kinase C (PKC) in peripheral nerve regeneration by using the cultured adult mouse sciatic nerve, which displays regrowth of sensory axons under serum-free conditions. By the use of immunohistochemistry we show that one of the isoforms of PKC, PKC beta, is present in the nerve cell bodies of normal nerves and is upregulated after injury. In spite of this, the specific PKC inhibitor chelerythrine at 5 microM, a concentration well above its IC50 value for PKC, failed to reduce the outgrowth distance of new axons. This was not due to impermeability of the drug, since the same concentration caused a clear reduction of the injury-induced proliferation of Schwann cells in the crush region. Likewise, HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinases, also lacked effect on outgrowth when used on its own, even at very high concentrations (100 microM). In contrast, outgrowth was significantly reduced when 5 microM chelerythrine and 5 microM HA-1004 were used in combination. In conclusion, the present results suggest that PKC-activity is important but not indispensable for the regeneration process. Successful completion of the latter could be achieved by several, perhaps redundant, phosphorylation systems.

Early regeneration in vitro of adult mouse sciatic axons is dependent on local protein synthesis but may not involve neurotrophins

The sensory axons of the adult mouse sciatic nerve were shown to regenerate after a local test crush lesion in vitro in a serum-free medium. The average outgrowth distance of the leading axons after culturing for 3 days was 2.8 +/- 0.1 mm, which was shorter than in vivo (3.8 +/- 0.2 mm). With the use of a compartmentalised culture system we could show that regeneration was partially dependent on local protein synthesis in the injury region. The initial stages of regeneration did not seem to involve neurotrophins since both K252a and K252b, selective and nontoxic inhibitors of the neurotrophin actions, failed to inhibit axonal growth. The present in vitro model system offers favourable conditions to investigate the early events of the regeneration process in an adult mammalian peripheral nerve.

Regeneration in vitro of the adult frog sciatic sensory axons

The adult frog sciatic nerve offers several advantages as an in vitro model to study nerve regeneration. The nerve with the attached dorsal root ganglia can easily be isolated and incubated in a culture medium for several days. If the nerve is subjected to a crush immediately after dissection there is a delay of 3.4 days after which the sensory axons start to regenerate into the distal nerve stump at a constant rate of about 1.1 mm · day-1 in serum-containing and 1.0 mm · day-1 in serum-free medium. Serum-free cultures may be used in future studies to examine the effect of various neurotrophic factors. The existence of an accurate method for examining the outgrowth distance, based on axonal transport of labelled proteins, contributes to the attractiveness of the model. A compartmental culture system permits separate exposure of the ganglia and the nerve to different agents. Taking advantage of this, pharmacological studies suggest that Schwann cells produce signals, dependent on newly transcribed RNA, which transform the preparation into a growth state. The present model system offers favourable conditions to learn more about the early events and also the subsequent steps of the regeneration process.

The role of ornithine decarboxylase and polyamines in regeneration of the frog sciatic nerve.

The current study examined both in vivo and in vitro the effects of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), on regeneration of sensory axons from a local crush of the adult frog sciatic nerve. If daily injections of DFMO started at the same time as crushing and continued throughout the regeneration period (7 days) the outgrowth in vivo of new sensory axons was reduced by about 30%. If DFMO injections started 2 days after crushing, the outgrowth distance did not differ from control values. The sensory axons of a cultured frog sciatic nerve with the attached spinal ganglia start to regenerate from a local crush applied 7 days after the start of the incubation. Five days after crushing the outgrowth distance was 4.5 mm. At the end of the culturing period (7 + 5 days) both the putrescine and spermidine concentrations in the ganglia had increased about 2.5 times, whereas the spermine concentration remained constant. The presence of 10 mM DFMO throughout the culturing period, 7 + 5 days, almost depleted putrescine and prevented the spermidine increase in the ganglia without affecting the regeneration distance. In the nerve putrescine was only reduced by 55% and the other polyamines were unaffected by DFMO. The results show that DFMO influences the early onset of regeneration in vivo. The in vitro results indicate that this is not due to a close mechanistic relationship between the perikaryonal ODC/polyamine system and nerve regeneration. The question of whether polyamines are of local importance for regeneration of the frog sciatic nerve cannot be answered by the present results.

Adenosine inhibition of the regeneration in vitro of adult frog sciatic sensory axons

The sensory axons of the adult frog sciatic nerve have earlier been shown to regenerate in vitro. If a local test crush is made at the initiation of culturing, regeneration starts after 3.4 days and proceeds at a rate of about 0.8-0.9 mm/day for several days. In the present experiments regeneration was inhibited by adenosine in a reversible and dose-dependent fashion. Similarly, both an adenosine analogue, 2-chloroadenosine (2-CA), and a non-hydrolyzable ATP analogue, AMP-PNP, reduced the outgrowth of sensory axons. The effect of adenosine was partially antagonized by theophylline at a critical concentration. Using a compartmental system, it could clearly be shown that adenosine exerted its effects at the outgrowth region. Adenosine, 2-CA, and AMP-PNP were also found to inhibit the proliferation of Schwann cells in the regenerating nerve. Various experiments showed that the latter can not explain the outgrowth inhibitory effects, which could be mediated by adenosine receptors associated with the elongating axons.

Growth cones of regenerating adult sciatic sensory axons release axonally transported proteins

Labelled, rapidly transported axonal proteins were shown to be released from adult frog sciatic sensory neurons, regenerating in vitro after a crush injury. The spatial distribution of the transported, released proteins could accurately be resolved by culturing the nerve on nitrocellulose paper, which trapped the released proteins. The release was located to the crush and to the entire outgrowth region. When regeneration was inhibited by adenosine, the release was limited to the crush site, implying that the release was linked to the growing axons. Other experiments suggested that the release emanated from growth cones. Furthermore, two-dimensional electrophoretical analysis of both fast axonally transported and of released proteins showed that the latter represented a selection of the transported protein species.

An improved HRP method for tracing axons in whole-mount preparations during early stages of regeneration in peripheral nerves of adult animals

An improved method using HRP for visualizing axons in whole-mount preparations is described. Application of this method to preparations of regenerating frog peripheral nerves maintained in organ culture was used to show that regeneration following nerve crush begins after about 3 days.